The process of phosphorylation, defined as the attachment of a phosphate moiety to a biological molecule through the action of enzymes called kinases, represents one course by which intracellular signals are propagated from molecule to molecule resulting finally in a cellular response. Within the cell, proteins can be phosphorylated on serine, threonine or tyrosine residues and the extent of phosphorylation is regulated by the opposing action of phosphatases, which remove the phosphate moieties. While the majority of protein phosphorylation within the cell is on serine and threonine residues, tyrosine phosphorylation is modulated to the greatest extent during oncogenic transformation and growth factor stimulation (Zhang, Crit. Rev. Biochem. Mol. Biol., 1998, 33, 1-52).
Because phosphorylation is such a ubiquitous process within cells and because cellular phenotypes are largely influenced by the activity of these pathways, it is currently believed that a number of disease states and/or disorders are a result of either aberrant activation or functional mutations in kinases and phosphatases. Consequently, considerable attention has been devoted recently to the characterization of tyrosine kinases and tyrosine phosphatases.
SHP-1 (also known as Src homology region 2-domain phosphatase, SHP, PTP1C, SHPTP1, HCP and PTPN6) is a cytosolic tyrosine phosphatase isolated by several different labs and shown to exist as two alternatively spliced forms in epithelial and hematopoietic tissues that are regulated by two tissue-specific promoters (Plutzky et al., Proc. Natl. Acad. Sci. U.S.A., 1992, 89, 1123-1127; Shen et al., Nature, 1991, 352, 736-739; Yi et al., Blood, 1991, 78, 2222-2228). The protein is predominantly expressed in hematopoietic cells and the gene is located in a region on chromosome 12 frequently involved in deletions associated with acute leukemia (Yi et al., Mol. Cell. Biol., 1992, 12, 836-846).
SHP-1 expression has been shown to be elevated in leukemic cells and this may be involved in their increased susceptibility to differentiation by phorbol ester (Kasugai et al., Cancer Lett. (Shannon, Irel.), 1997, 120, 223-227; Uchida et al., J. Biol. Chem., 1993, 268, 11845-11850). In these studies, the use of antisense oligonucleotides targeting the first and second AUG codons of SHP-1 were used to decrease the PMA-induced adherence of a variant human leukemia cell line suggesting that the high expression of the SHP-1 phosphatase in these cells was related to the differentiation process (Kasugai et al., Cancer Lett. (Shannon, Irel.), 1997, 120, 223-227).
Mice lacking the SHP-1 locus have been shown to exhibit severe defects in immune and hematological function, most of which are attributed to abnormalities in the interaction between lymphoid and myeloid cell lineages (Shultz et al., Trends Biotechnol., 1997, 15, 302-307).
The SHP-1 protein contains two src homology 2 (SH2) domains, conserved regions of approximately 100 amino acids originally identified in Src protein tyrosine kinases, that promote protein--protein interactions through phosphotryosyl residue binding (Neel, Semin. Cell. Biol., 1993, 4, 419-432). These two domains have been shown to display differential functions in the regulation of the SHP-1 phosphatase and consequently affect different signaling pathways. The N-terminal SH2 domain serves as a regulatory and recruiting domain, producing an autoinhibitory effect through intramolecular interactions with the internal catalytic phosphatase domain. While the C-terminal SH2 domain acts merely to recruit other proteins for intermolecular interactions necessary for signal transduction (Pei et al., Proc. Natl. Acad. Sci. U.S.A., 1996, 93, 1141-1145).
The SHP-1 protein interacts with several different types of signaling molecules within the cell through the SH2 domains. These include the .beta.c subunit of the human interleukin 3 receptor (Bone et al., J. Biol. Chem., 1997, 272, 14470-14476), the high affinity IgE receptor (Kimura et al., J. Immunol., 1997, 159, 4426-4434), and killer cell inhibitory receptors (Olcese et al., J. Immunol., 1996, 156, 4531-4534). It has also been shown to interact with biliary glycoprotein, an immunoglobulin like cytohesion molecule with tumor suppressor function, in epithelial cells (Huber et al., J. Biol. Chem., 1999, 274, 335-344).
SHP-1 also plays a critical role in B and T cell signaling (Siminovitch and Neel, Semin. Immunol., 1998, 10, 329-347). By interacting with SLP-76, an SH2 domain containing leukocyte protein, SHP-1 negatively regulates B cell antigen receptor signaling in B cells (Mizuno et al., J. Exp. Med., 1996, 184, 457-463). In T cells, SHP-1 has been shown to negatively regulate T cell response to antigen receptor stimulation by participating in the Ras/MAPK response pathway (Pani et al., J. Exp. Med., 1996, 184, 839-852). The SHP-1 phosphatase has also been associated with G-protein signaling as it is phosphorylated by kinases downstream of G-protein .beta..gamma. subunit linked receptors (Gaits et al., J. Biol. Chem., 1996, 271, 20151-20155). Finally, in MCF-7, a breast adenocarcinoma cell line, it was shown that the translocation of SHP-1 could be induced by an octapeptide analog of somatostatin in a G-protein dependent manner (Srikant and Shen, Endocrinology, 1996, 137, 3461-3468). Disclosed in U.S. Pat. No. 5,659,012 is a peptide inhibitor of SHP-1 that binds to the src homology 2 domains of the protein (Klingmuller et al., 1997).
In non-hematopoetic cells, SHP-1 has been shown to play a role in multiple pathways as both a positive and negative regulator of signal transduction. In 293 cells, overexpression of a catalytically inactive mutant of SHP-1 suppressed the effects of epidermal growth factor (Su et al., J. Biol. Chem., 1996, 271, 10385-10390).
To date, strategies aimed at modulating SHP-1 function have involved the use of antibodies, molecules that affect the enzymatic activity the protein, antisense molecules and gene knock-outs in mice. However, there are no known therapeutic agents which effectively inhibit the synthesis of SHP-1. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting SHP-1 function.
Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of SHP-1 expression.